Dear colleagues/scientists,
You may know we can do a back calculation to get a rough measurement of the concentration of the PCR product we amplified in comparison of both ladder and PCR product intensities. According to the manual.
If we load 4 µL of both ladder and the PCR sample; think roughly our PCR band locates close to 400 bp fragment size. Intensity of our PCR product greater than 2 folds than that of the 400bp ladder fragment, therefore we can roughly say quantity of our PCR product is about 80 ng (According to the manual I have provided).
However my question is can't we do this quantification using Nanodrop Spectrophotometer?
If can what is the blanking solution we have to consider? (If we use Go Taq ® Green master mixture for the above amplification)
You can but for that you have to clean the PCR product.
Since in a PCR reaction, in addition to amplified fragment there are also non-amplified DNA, primers, salts, buffer etc. which interfere the correct quantification.
Abhijeet Singh, Thank you very for your reply, If we purify in someway, What would be the best purification technique and in that case what would be the blanking solution in spectrophotometric analysis.
There are many kits available for the purpose. You can also purify the product by magnetic bead separation.
In any case the blank should be same as the elution solution.
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